Historically, the aryl hydrocarbon receptor (AhR) has been studied for its transcriptional regulation of genes encoding cytochrome P450 enzymes, which metabolize environmental and endogenous substrates into toxic and mutagenic intermediates. Accumulating studies support the hypothesis that the AhR also plays an important role in malignant epithelial cell growth and invasion apart from its role in formation of mutagens and in the absence of environmental chemicals. This new paradigm is based on several key observations: 1) AhR expression is increased dramatically in carcinogen-induced rat and mouse mammary tumors and in "spontaneous" human mammary tumor lines. 2) Constitutive AhR activation is indicated by nuclear AhR localization in rat, mouse, and human mammary tumors and by AhR binding to gene promoters in the absence of environmental chemicals. 3) Constitutively active AhR regulates the expression of multiple genes, including CYP1B1, CK21, and Slug, a master regulator of tumor invasion. 4) Recent studies suggest that increased AhR activity in mammary tumors also contributes to cell migration and invasiveness. 5) Molecular downregulation of the AhR suppresses breast cancer cell proliferation and reverts cells to a non-aggressive phenotype. Molecular and biologic strategies have provided significant evidence that the AhR participates, beyond mutagenesis, in multiple mechanisms that contribute to tumor formation, growth and invasion. Therefore, we can exploit our ability to examine effects of constitutively active AhR to determine how chemical antagonism of the AhR may translate into breast cancer prevention or a therapeutic approach to suppress tumor progression. Thus, we propose a new hypothesis: Targeting the constitutively active AhR with naturally occurring, non-toxic antagonists represents a feasible therapeutic approach to inhibit breast tumor growth and invasion. Three specific aims are proposed: 1. Investigate strategies to maximize antagonism of the AhR by examining the potential for synergistic interaction in mixtures of antagonists, performing a high-throughput screen for novel, potent antagonists from natural product extract libraries (NCI Natural Products Repository) and examining the "chemical knockout" approach for improving AhR inactivation. 2. Define the molecular mechanisms of chemical antagonism of the constitutively active AhR in a breast cancer model by establishing antagonist effects on AhR transactivation of endogenous gene expression and examining antagonist-mediated changes in AhR-DNA interactions. 3. Establish the functional consequences of chemically antagonizing the constitutively active AhR using optimal AhR antagonists. The translational impact of these studies lies in the ability of known and newly identified antagonists to suppress tumor growth and invasion. Here, potentially therapeutic AhR antagonists will be evaluated for their ability to block the biological outcomes of constitutive AhR activity in human mammary tumor cell lines. Collectively, these studies will provide the foundation for preclinical studies on the potential for potent AhR antagonists to prevent and/or treat breast cancer in vivo. PUBLIC HEALTH RELEVANCE: We hypothesize that the hyper-expression of a protein, called the aryl hydrocarbon receptor, and its binding to DNA contributes to the growth and progression of breast tumors. Here we propose that chemicals that impede the function of this receptor (i.e. antagonists) will be effective at downregulating this protein's activity and therefore will suppress breast tumor growth and metastasis. Screening of plant and marine natural product libraries will provide a source of novel antagonists that can be tested for their interaction with this receptor and their mechanism of interference with tumor growth, ultimately resulting in the development of therapeutic agents for the treatment of breast cancer.